1. Field of the Invention
This invention relates to methods and apparatus for preparing red blood cell substitute products, i.e., hemoglobin products. It further relates to an acellular red blood cell substitute comprising an essentially tetramer-free, cross linked, polymerized, pyridoxylated hemoglobin solution which is free of stromal contaminants.
2. Description of Related Art
For a number of years, blood banks have provided whole blood for replacement during surgery, because of trauma, or for other situations. However, whole blood obtained from human donors is not suitable for a variety of uses. In particular, the use of whole blood is problematic because of the requirement for donor-typing, stability and shelf-life problems and toxicity caused by viruses and other contaminants. These problems are especially pertinent to emergency situations, such as the use of blood by the military. Consequently, much effort has been devoted to the development of substitutes for whole blood obtained from human donors. This development has resulted in various modifications to blood from human or other mammalian sources. Stroma-free hemoglobin is known in the art to have oxygen transport and reversible oxygen (or ligand) binding capacities. Since toxicity problems have precluded use as a blood substitute, stroma-free hemoglobin has required further modifications to provide a nontoxic, useful pharmaceutical product.
These modifications include (1) rendering hemoglobin free or substantially free of stroma and stromal contaminants; (2) pyridoxylation; (3) polymerization or cross-linking; (4) removal of tetramer, and (5) modification with carbon monoxide or other ligands.
However, hemoglobin solutions prepared by these techniques, while capable of carrying sufficient quantities of oxygen to support life, have been plagued with many undesirable side effects and properties. For example, a major troubling side effect is a decrease in kidney performance. These changes were thought to be due to the presence of unwanted contaminants such as bacterial endotoxin or fragments of red cell membranes (stroma). While contaminants such as these can indeed produce renal alterations, hemoglobin solutions essentially free of the above contaminants still produce substantial renal dysfunction. The cause for the renal dysfunction has been ascribed to physiologically unacceptable amounts of unpolymerized hemoglobin tetramer. Other undesirable side effects of the infusion of tetrameric hemoglobin are vasoconstriction, hemoglobinuria, depression of heart rate, elevation of mean arterial blood pressure and extravasation of infusate especially into the peritoneal cavity.
In practice, no known hemoglobin-derived blood substitute has been successful in totally avoiding toxicity problems. These products also have unacceptably low half-lives after administration to human patients. Such half-lives require replacement of blood volume repeatedly over short periods of time. Consequently, there is a substantial need for hemoglobin products that are non-toxic to patients and have substantial half-lives after administration. Of course, these products must be capable of reversibly transporting oxygen to tissues in a manner similar to that achieved by whole blood.
The present invention provides hemoglobin substitutes that are non-toxic to humans and have substantial half-lives of at least 15 hours when administered to humans. The hemoglobin products of the invention are stroma-free, pyridoxylated and polymerized as well as being free of viral and other toxic contaminants. Further, these products are substantially free of leukocytes (white blood cells) and platelets.
The present invention also encompasses processes for preparing the inventive hemoglobin substitutes. The processes include removing leukocytes and platelets from blood; washing and lysing the red blood cells; removing stromal contaminants and stroma by filtration and heat-treating; preparing the deoxy form of the hemoglobin; pyridoxylation and polymerization; further purification and concentration; and deoxygenation. The resulting hemoglobin product may be then formulated to provide a hemoglobin product having levels of various electrolytes within normal physiological ranges.
The invention also provides an aqueous formulation of pyridoxylated, polymerized hemoglobin, where the hemoglobin is a glutaraldehyde-polymerized hemoglobin containing tetrameric material, having the molecular weight profile of FIG. 3. This formulation may be used to prepare an acellular red blood cell substitute. In this aspect, the formulation is first purified to remove tetramer and then combined with appropriate amounts of electrolytes to produce a physiologically acceptable, acellular, red blood cell substitute that may subsequently be used to treat a human patient requiring an infusion of an oxygen carrier.